AIDS was first reported in the United States in 1981 and has since become a major worldwide pandemic. AIDS is caused by the human immunodeficiency virus, or HIV. Today more than 30 million people living throughout the world are infected by the virus (Cohen, Hellmann et al. 2008). HIV progressively destroys the body's ability to fight infections and other diseases by killing or damaging cells of the body's immune system, specifically eliminating immune cells that express the CD4 molecule, such as CD4+ helper T-lymphocytes (leading to an inverted CD4/CD8 T-cells ratio) and cells of the monocyte/macrophage lineage (Fauci 1996).
CD4 T-cells mature into two polarized functional types, called Th1 and Th2 (Mosmann and Coffman 1989; Mosmann and Sad 1996). Th1 CD4+ cells are responsible for mediating cellular immunity and Th2 CD4+ cells are responsible for mediating humoral immunity (D'Elios and Del Prete 1998). HIV infection causes a gradual loss of the Th1 subset resulting in an inverted Th1/Th2 ratio (Becker 2004) and loss of cellular immunity. The loss of Th1 immunity and switch to Th2-dominated immunity in HIV patients has been correlated with profound immunosuppression and the progression from HIV positive status to AIDS (Klein, Dobmeyer et al. 1997). One of the leading causes of death of patients with AIDS is opportunistic infections due to the suppression of the cellular immune system (Baker and Leigh 1991).
HIV has multiple strategies for immune evasion. These strategies include mutational escape, latency, masking of antibody-binding sites on the viral envelope, down-modulation of the class I major histocompatibility complex (MHC-I), up-regulation of the Fas ligand on the surface of infected cells (Piguet and Trono 2001) and inducing the production of IL-10 (Leghmari, Bennasser et al. 2008; Brockman, Kwon et al. 2009). In addition, Some viral genes such as vif, vpr, vpu, and nef genes translate proteins that act to suppress anti-viral immune responses (Kirchhoff 2010). These viral escape mechanisms make the virus elusive for control using immunological methods (Migueles, Tilton et al. 2006; Bansal, Yue et al. 2007; Feinberg and Ahmed 2012; Teshome and Assefa 2014).
HIV virology has been intensively studied and the viral structure and life cycle of HIV has been described (Pomerantz 2002; Sierra, Kupfer et al. 2005; Li and Craigie 2006; Cohen 2008; Scherer, Douek et al. 2008; Fanales-Belasio, Raimondo et al. 2010). A single HIV particle is called a virion. The virion is shaped like a spiked sphere. The central core of the sphere is called the capsid. The capsid contains two single strands of HIV RNA called viral RNA. When viral RNA is detected in the serum, the quantity of viral RNA is called the viral load. The viral RNA codes for three enzymes important to the virus's life cycle called reverse transcriptase, integrase, and protease. These enzymes are foreign to human immune system and are capable of being recognized by CD8+ CTL killer cells (Haas, Samri et al. 1998). In this manner, cells that express these viral enzymes are targets for immune elimination. However, the viral RNA also contains instructions for production of viral accessory proteins that serve to assist the virus to evade immune elimination (Seelamgari, Maddukuri et al. 2004; Malim and Emerman 2008).
Surrounding the core is a protective lipid (fat) bilayer which forms a shell around the capsid (Frankel 1996; Bradbury 2013). This shell is called the viral envelope. Embedded within the viral envelope is a HIV protein called env. The env protein is made up of two glycoproteins, gp120 and gp41, that protrude from the virion forming the spikes. The cap of the spike is gp120 and the stem is gp41. For HIV to enter a host cell, it must first use gp120 to attach to a CD4 receptor (Pancera, Majeed et al. 2010; Guttman and Lee 2013).
After gp120 successfully attaches to a CD4 cell, the molecule can change shape to avoid recognition by neutralizing antibodies, a process known as conformational masking (Kwong, Doyle et al. 2002). The conformational change in gp120 allows it to bind to a second receptor on the CD4 cell surface called a chemokine receptor.
The chemokine receptor on the CD4 cells surface used as a co-receptor for the HIV virion is either CCR5 or CXCR4 (Moore, Trkola et al. 1997). The viral preference for using one chemokine co-receptor versus another is called ‘viral tropism’. Chemokine receptor 5 (CCR5), is used by macrophage-tropic (M-tropic) HIV to bind to a cell (Cohen, Kinter et al. 1997). About 90% of all HIV infections involve the M-tropic HIV strain. CXCR4, also called fusin, is a chemokine receptor used by T-tropic HIV (ones that preferentially infect CD4 T-cells) to attach to the host cell (Hoxie, LaBranche et al. 1998). Another co-receptor called DC-SIGN is expressed on dendritic cells and also binds gp120 in order to facilitate viral infection of these important cells involved in cellular immunity (Cunningham, Harman et al. 2007). Viral infected macrophages can interact with CD4 T-cells and pass the virus through cell-to-cell contract (Martin and Sattentau 2009; Poli 2013). In addition, HIV can induce T-cells to form syncytium to facilitate cell-to-cell viral transfer (Emilie, Maillot et al. 1990; Kozal, Ramachandran et al. 1994; Margolis, Glushakova et al. 1995).
Transmission of HIV results in the establishment of a new infection, starting from even a single virion particle. HIV virons are replicated within host infected cells and released into the plasma which causes viremia and persistent infection of immune cells in all of the lymphoid tissues in the body. HIV preferentially infects T cells with high levels of CD4 surface expression and those subsets of T cells that co-express CCR5. The subset of memory T-cells are a preferred target (Helbert, Walter et al. 1997), particularly HIV-specific memory T cells (Douek, Brenchley et al. 2002) and Th2/Th0 cells (Maggi, Mazzetti et al. 1994).
With the onset of immunodeficiency, the virus evolves to infect new cell types. This correlates with a tropism change involves switching from preference for CCR5 co-receptor to the alternative CXCR4 co-receptors. This switch corresponds with an expansion of infected cells to include naive CD4+ T cells in addition to the preferred memory cells. Similarly, the virus evolves the ability to enter cells with low levels of CD4 on the surface and this potentiates the ability to infect monocyte/macrophages. Naive cells are found almost exclusively in the secondary lymphoid organs, while memory cells and macrophages have a much wider tissue distribution, including the brain, tissue and organ systems. Infection of naive cells and macrophages establishes pools of viral infected cells throughout the body and in locations that are difficult to target with drugs or immunotherapy.
M-tropic and T-tropic strains of HIV can also coexist in the body, further complicating the ability to target elimination of the virus. At some point in infection, gp120 is able to attach to either CCR5 or CXCR4. A HIV virion with this property is called a dual tropic virus or R5X4 HIV (Toma, Whitcomb et al. 2010; Loftin, Kienzle et al. 2011; Svicher, Balestra et al. 2011). HIV that can utilize the CXCR4 receptor on both macrophages and T-cells is also termed dual-tropic X4 HIV (Huang, Eshleman et al. 2009; Gouwy, Struyf et al. 2011; Xiang, Pacheco et al. 2013). Mixed tropism results when an individual has two virus populations; one using CCR5 and the other CXCR4 to bind to the CD4 T-cell. Since the virological behavior of T-tropic and M-tropic viruses vary, mixed tropism creates a difficult problem for drug design.
Once the HIV envelope has attached to the CD4 molecule and is bound to a co-receptor, the HIV envelope utilizes a structural change in the gp41 envelope protein to fuse with the cell membrane and evade neutralizing antibodies (Chen, Kwon et al. 2009). The HIV virion is then able to penetrate the target cell membrane.
Once within a host cell, the viral enzyme reverse transcriptase converts the viral RNA to viral DNA. Reverse transcriptase inhibitors are developed as an anti-HIV therapy (Nurutdinova and Overton 2009; Chowers, Gottesman et al. 2010; Zhan and Liu 2011). Once the viral RNA is transcribed to DNA, the DNA is then able to enter the nucleus of the host cell. Using another viral enzyme called integrase, the viral DNA is able to integrate into the host cell's chromosomal DNA. Integrase inhibition is another target of anti-viral drug development (Geretti, Armenia et al. 2012; Okello, Nishonov et al. 2013). The integrated viral DNA is called provirus and is replicated along with the host chromosome when the host cell divides. The integration of provirus into the host DNA provides the latency that enables the virus to effectively evade host immune responses.
When the host cell is activated to divide, production of viral proteins and viral RNA takes place as the provirus is transcribed along with the host DNA. Viral proteins are then assembled using the host cell's protein-making machinery. The virus's protease enzyme allows for the processing of newly translated viral polypeptides into the proteins which constitute the virus. These various proteins are then ultimately assembled into viral particles. Protease inhibitors are another class of anti-viral drugs for treatment of HIV infection (Wattanutchariya, Sirisanthana et al. 2013). The assembled virus uses the nuclear capsid protein called gag to interact with host protein machinery to cause the budding of the virus and release of whole virus from the host cell (Dussupt, Javid et al. 2009). Alternatively, the budding HIV can transfer directly from cell-to-cell interaction (Fais, Capobianchi et al. 1995). Many viral particles can bud from of a single cell over the course of time, eventually lysing the cell membrane killing the cell.
Cells actively producing virus are vulnerable to attack by CD8 cells (cytotoxic T-lymphocytes or CTLs). CTL cells require help from Th1 CD4 cells to kill cells that are producing virus (Wodarz 2001). In HIV infection, the viral load can be kept in a steady state with the rate of immune-mediated destruction of viral producing cells balanced with the rate of release of viral particles from infected cells. In this steady state, the viral load is maintained at a set point level (Korthals Altes, Ribeiro et al. 2003; Kaul, MacDonald et al. 2010). When CD4 counts drop sufficient to lose this helper function for CTL, the set point control is lost and the viral load climbs. Eventually this leads to a fall in CD4 counts, loss of cellular immunity and eventually leading to AIDS. An HIV infection can be in such a steady state for eight to ten years before the clinical syndrome of AIDS occurs (Jurriaans and Goudsmit 1996; Callaway and Perelson 2002; Maenetje, Riou et al. 2010).
The most obvious laboratory observation in HIV infection is a decline in the number of CD4+ T− cells found in the blood and a decline in the CD4/CD8 ratio. Increase in viral load (viral RNA) can be detected by sensitive PCR tests.
Highly active antiretroviral therapy (HAART) for the chronic suppression of HIV replication has been the major accomplishment in HIV/AIDS medicine. HAART cocktails contain drugs with different mechanisms of action designed to block the natural virus life cycle at different points. For example, HAART can contain reverse transcriptase, integrase, protease and binding (Carter 2003; Laurence 2004; 2007) inhibitors. Many patients are now in their second decade of treatment, with levels of plasma HIV RNA (viral load) below the limits of detection of clinical assays (e.g., <50 copies/ml). New HAART drugs are being developed to interfere with the viral life cycle. For example, since CCR5 has been identified as a major HIV co-receptor this has lead to the development of drugs that target the virus-CCR5 interaction, including the first-in-class approved drug, Maraviroc (Rusconi, Vitiello et al. 2013).
Since HAART is not able to completely eliminate the virus, life-long antiviral therapy is needed to control HIV infection. Such therapy is expensive and prone to drug resistance, cumulative side effects and unknown effects of long-term treatment. HAART has several long-term side effects including kidney, liver, and pancreatic problems; and changes in fat metabolism, which result in elevated cholesterol and triglyceride levels and an increased risk for strokes and heart attacks (Carter 2003; Laurence 2004; 2007). In addition, some viruses have evolved resistance to HAART (Fumero and Podzamczer 2003; Tebit, Sangare et al. 2008; Loulergue, Delaugerre et al. 2011).
HIV infection persists in spite of efficacious HAART therapies as evidenced by rapid rebound of viremia upon cessation of HAART therapy most often within 3-10 days (Neumann, Tubiana et al. 1999; Van Gulck, Heyndrickx et al. 2011). This phenomenon is thought to be due to the early establishment of a stable reservoir of latently infected cells with integrated viral DNA that seeds the production of virions after HAART cessation.
The goal of HAART therapy in HIV-infected patients is to reduce plasma HIV viral load (HIV RNA) to undetectable levels and to increase the CD4 cell count. Achievement of this goal reduces the rate of disease progression and death. However, some patients experience isolated episodes of transiently detectable HIV RNA or viral rebound (Staszewski, Miller et al. 1998; Butler, Gavin et al. 2014). The causes of viral rebound are still unclear. Rates of viral rebound of 25-53% have been reported among patients on HAART who have achieved undetectable HIV RNA. Viral rebound that then persists as a low level viremia (set point level) may lead to genetic mutations in the virus leading to drug resistance.
Patients with persistent low-level viremia have a higher rate of virological failure. Persistent low-level viremia is defined as plasma HIV RNA levels in the range of 51-1000 copies/mL for at least 3 months and on at least two consecutive clinic visits. Virological failure is defined as two consecutive plasma HIV RNA levels >1000 copies/mL.
After HAART initiation, most patients experience improved immune function and maintain viral suppression; however, there remains a subset of patients who have suboptimal immunologic responses—defined as the failure to achieve and maintain an adequate CD4 response despite use of HAART therapy. Patients with inadequate CD4 counts on HAART therapy are said to have immunological failure. Adequate CD4 counts are generally defined as >500 cells/mm3 over a specific period of time (e.g., 4 to 7 years). Immunological failure increases the risk of AIDS- and non-AIDS-related morbidity and mortality. For example, a low CD4 count of <500 is associated with an increased risk in cardiovascular, hepatic, and renal disease and cancer.
Cytotoxic T lymphocyte (CTL) and Natural Killer (NK) cell responses are important to the initial decrease in HIV viral load seen in the first several months after acute infection (Borrow, Lewicki et al. 1997; Fan, Huang et al. 1997; Smalls-Mantey, Connors et al. 2013). These beneficial cellular immune responses diminish with disease progression and cannot be recovered with antiretroviral therapy alone. CTL responses generally require CD4 cell help to be effective (Wodarz 2001).
Recent studies suggest a therapeutic vaccine may help to restore cellular immunity and CTL and NK responses to the virus. Therapeutic HIV vaccines are designed to control HIV infection by boosting the body's natural immune response. HIV-specific T-cell-based vaccines have been extensively studied in both prevention and therapeutic settings, with most studies failing to show benefit, and some suggesting harm (Papagno, Alter et al. 2011). There are currently no FDA-approved therapeutic HIV vaccines.
So far it has been impossible to cure HIV despite long-term viral suppression on HAART. The rapid rebound despite powerful viral suppression and blockage of viral entry is thought to be due to the reservoirs of latently infected cells unaffected by viral suppression and unable to be targeted for immune elimination, also the continuous sub-clinical viral production from some cells in lymph nodes and tissues and the ability of the virus to spread through cell-to-cell contact as an alternative to entry pathway all serve to maintain viral persistence.
While there are descriptions of some patients that can remain with undetectable virus without HAART, these so called “secondary controllers” are infected with less infectious types of HIV (Lobritz, Lassen et al. 2011; Van Gulck, Bracke et al. 2012). For the majority of patients, HAART is a lifetime requirement for disease control.
The only report of long-term viral suppression after cessation of HAART therapy is the so called “Berlin Patient”. The Berlin Patient received an allogeneic stem cell transplant for treatment of his leukemia. The donor had a special genetic characteristic (two copies of the recessive CCR5Δ32 allele) which results in the inability to express the CCR5 receptor on the surface of CD4 cells. Thus the donor cells for the transplant were resistant to viral entry. After transplant, the patient was able to stop all HAART anti-retroviral therapy and remained with undetectable viral load for 3½ years after the transplant (Hutter, Nowak et al. 2009).
It is possible that innate or acquired immunity delivered by the donor immune system may have contributed to the elimination of cells with active HIV replication. The patient experienced graft versus host disease (GVHD), and it is possible that an allogeneic immune response directed against host lymphocytes had a purging effect on the latent HIV reservoir in lymphocytes.
Allogeneic stem cell transplant is a highly toxic procedure with high treatment related mortality and morbidity. The high toxicity is related to the need for chemotherapy conditioning regimes and to the often lethal GVHD side-effect. The toxicity of GVHD limits the clinical use of allogeneic transplant procedures to terminally-ill patients without other treatment options. However, in HIV+ patients that are stable on HAART medication, it is not clinically feasible to treat with allogeneic stem cell transplant.
Further, allogeneic transplant requires HLA tissue matched donors. Only ⅓ of individuals have a related HLA-matched donor and fewer are able to find an unrelated HLA matched donor. Moreover, even if a matched donor can be identified, the donor must be homozygous for the CCR5Δ32 mutation, which is an extremely rare genetic phenotype (Jiang, Wang et al. 1999; Williamson, Loubser et al. 2000). Thus the lack of suitable donors and the toxicity of allogeneic transplant procedures makes it impossible to translate data from the Berlin patient to benefit the majority of HIV infected patients.
Accordingly, additional non-toxic therapies are needed in order to exploit the mechanisms that enabled the Berlin Patient to enjoy long-term HAART cessation. In addition, treatment options for virologic failure and immunological failure while on HAART treatment are urgently needed.